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Human cell line models have been widely used for testing of novel anticancer compounds and for predicting clinical response to monotherapies and combinatorial therapies. For many years, standard monolayer culture conditions were used as gold standard, only surpassed by in vivo testing of mouse models. Recently, the incorporation of three-dimensional culture has been shown to further improve predictive compound testing. In view of the renewed interest in anti-RAS cancer therapy, we provide a protocol for establishing colorectal cancer organoids which are characterized by a high prevalence of KRAS mutations.

Circadian rhythms are essential to the temporal regulation of molecular processes in living systems and as such to life itself. Deregulation of these rhythms leads to failures in biological processes and eventually to the manifestation of pathological phenotypes including cancer. To address the questions as to what are the elicitors of a disrupted clock in cancer, we applied a systems biology approach to correlate experimental, bioinformatics and modelling data from several cell line models for colorectal and skin cancer. We found strong and weak circadian oscillators within the same type of cancer and identified a set of genes, which allows the discrimination between the two oscillator-types. Among those genes are IFNGR2, PITX2, RFWD2, PPARγ, LOXL2, Rab6 and SPARC, all involved in cancer-related pathways. Using a bioinformatics approach, we extended the core-clock network and present its interconnection to the discriminative set of genes. Interestingly, such gene signatures link the clock to oncogenic pathways like the RAS/MAPK pathway. To investigate the potential impact of the RAS/MAPK pathway - a major driver of colorectal carcinogenesis - on the circadian clock, we used a computational model which predicted that perturbation of BMAL1-mediated transcription can generate the circadian phenotypes similar to those observed in metastatic cell lines. Using an inducible RAS expression system, we show that overexpression of RAS disrupts the circadian clock and leads to an increase of the circadian period while RAS inhibition causes a shortening of period length, as predicted by our mathematical simulations. Together, our data demonstrate that perturbations induced by a single oncogene are sufficient to deregulate the mammalian circadian clock.

Because of the low overall response rates of 10-47% to targeted cancer therapeutics, there is an increasing need for predictive biomarkers. We aimed to identify genes predicting response to five already approved tyrosine kinase inhibitors. We tested 45 cancer cell lines for sensitivity to sunitinib, erlotinib, lapatinib, sorafenib and gefitinib at the clinically administered doses. A resistance matrix was determined, and gene expression profiles of the subsets of resistant vs. sensitive cell lines were compared. Triplicate gene expression signatures were obtained from the caArray project. Significance analysis of microarrays and rank products were applied for feature selection. Ninety-five genes were also measured by RT-PCR. In case of four sunitinib resistance associated genes, the results were validated in clinical samples by immunohistochemistry. A list of 63 top genes associated with resistance against the five tyrosine kinase inhibitors was identified. Quantitative RT-PCR analysis confirmed 45 of 63 genes identified by microarray analysis. Only two genes (ANXA3 and RAB25) were related to sensitivity against more than three inhibitors. The immunohistochemical analysis of sunitinib-treated metastatic renal cell carcinomas confirmed the correlation between RAB17, LGALS8, and EPCAM and overall survival. In summary, we determined predictive biomarkers for five tyrosine kinase inhibitors, and validated sunitinib resistance biomarkers by immunohistochemistry in an independent patient cohort.

RAS mutations are highly relevant for progression and therapy response of human tumours, but the genetic network that ultimately executes the oncogenic effects is poorly understood. Here, we used a reverse-engineering approach in an ovarian cancer model to reconstruct KRAS oncogene-dependent cytoplasmic and transcriptional networks from perturbation experiments based on gene silencing and pathway inhibitor treatments. We measured mRNA and protein levels in manipulated cells by microarray, RT-PCR and western blot analysis, respectively. The reconstructed model revealed complex interactions among the transcriptional and cytoplasmic components, some of which were confirmed by double pertubation experiments. Interestingly, the transcription factors decomposed into two hierarchically arranged groups. To validate the model predictions, we analysed growth parameters and transcriptional deregulation in the KRAS-transformed epithelial cells. As predicted by the model, we found two functional groups among the selected transcription factors. The experiments thus confirmed the predicted hierarchical transcription factor regulation and showed that the hierarchy manifests itself in downstream gene expression patterns and phenotype.

Transcriptome analysis of cancer cells has developed into a standard procedure to elucidate multiple features of the malignant process and to link gene expression to clinical properties. Gene expression profiling based on microarrays provides essentially correlative information and needs to be transferred to the functional level in order to understand the activity and contribution of individual genes or sets of genes as elements of the gene signature. To date, there exist significant gaps in the functional understanding of gene expression profiles. Moreover, the processes that drive the profound transcriptional alterations that characterize cancer cells remain mainly elusive. We have used pathway-restricted gene expression profiles derived from RAS oncogene-transformed cells and from RAS-expressing cancer cells to identify regulators downstream of the MAPK pathway.We describe the role of epigenetic regulation exemplified by the control of several immune genes in generic cell lines and colorectal cancer cells, particularly the functional interaction between signaling and DNA methylation. Moreover, we assess the role of the architectural transcription factor high mobility AT-hook 2 (HMGA2) as a regulator of the RAS-responsive transcriptome in ovarian epithelial cells. Finally, we describe an integrated approach combining pathway interference in colorectal cancer cells, gene expression profiling and computational analysis of regulatory elements of deregulated target genes. This strategy resulted in the identification of Y-box binding protein 1 (YBX1) as a regulator of MAPK-dependent proliferation and gene expression. The implications for a therapeutic application of HMGA2 gene silencing and the role of YBX1 as a prognostic factor are discussed.

The small GTP-binding proteins HRAS, KRAS and NRAS belong to a family of oncoproteins associated with many types of human cancer. Signal transduction processes initiated at receptor tyrosine kinases converge on RAS proteins which serve as molecular switches linking upstream signals with the transcriptional machinery. RAS proteins interact with a number of effector proteins that in turn activate the Raf/MEK/ERK pathway, the PI3K/PKB/Akt pathway, the RalGDS/Ral pathway and other downstream pathways. Mutations in RAS lock the protein in its active form. Chronic activation of the KRAS isoform is the basis for resistance toward antibody therapies targeting receptor tyrosine kinases, as an upstream stimulus through growth factor receptor-mediated activation is no longer required. However, the complexity of the RAS signaling system necessitates the search for additional activating mechanisms as well as biomarkers associated with pathway activation. During recent years, several RAS pathway-related gene signatures were identified, mostly by microarray-based gene expression profiling of normal versus RAS-transformed cells. The signatures can serve as a source of common biomarkers indicating functionally relevant downstream effects of the RAS signaling system. In searching for new markers, we compared the gene expression signatures compiled in 24 independent studies. We analyzed differentially regulated genes recovered in microarray studies on human specimens to discriminate paired normal and tumor tissues. Although the overlap between individual studies was low, this meta-analysis revealed Kruppel-like factor 5 (KLF5), the CD44 antigen and members of the epidermal growth factor (EGR)-family as common downstream effectors of RAS.

Most malignant features of cancer cells are triggered by activated oncogenes and the loss of tumor suppressors due to mutation or epigenetic inactivation. It is still unclear, to what extend the escape of emerging cancer cells from recognition and elimination by the immune system is determined by similar mechanisms. We compared the transcriptomes of HCT116 colorectal cancer cells deficient in DNA methyltransferases (DNMTs) and of cells, in which the RAS pathway as the major growth-promoting signaling system is blocked by inhibition of MAPK. We identified the MHC Class I genes HLA-A1/A2 and the ULBP2 gene encoding 1 of the 8 known ligands of the activating NK receptor NKG2D among a cluster of immune genes up-regulated under the conditions of both DNMT-deficiency and MEK-inhibition. Bisulphite sequencing analyses of HCT116 with DNMT deficiency or after MEK-inhibition showed that de-methylation of the ULPB2 promoter correlated with its enhanced surface expression. The HLA-A promoters were not methylated indicating that components of the HLA assembly machinery were also suppressed in DNMT-deficient and MEK-inhibited cells. Increased HLA-A2 surface expression was correlated with enhanced recognition and lysis by A2-specific CTL. On the contrary, elevated ULBP2 expression was not reflected by enhanced recognition and lysis by NK cells. Cosuppression of HLA Class I and NKG2D ligands and genes encoding peptide transporters or proteasomal genes mediates a strong functional link between RAS activation, DNMT activity and disruption of the antigen presenting system controlling immune recognition in colorectal cancer cells.

Many signal transduction processes converge on Ras proteins that serve as molecular switches to couple external stimuli with cytoplasmic and nuclear targets. Oncogenic mutations lock Ras proteins in their activated state. Cellular responses to permanent Ras activation such as the induction of neoplastic phenotypes are mediated by distinct transcriptional alterations. A number of studies have reported alterations of the genetic program because of short-term or long-term activation of Ras signaling pathways. However, a consistent pattern of Ras-related transcriptional alterations has not yet emerged, because currently available investigations were based on different methods for assessing mRNA expression profiles, on different types of cells, and on heterogeneous experimental conditions. Here we describe the "Ras signaling target array" (RASTA) representing approximately 300 Ras-responsive target genes. This customized oligonucleotide array is a universal tool for assessing transcriptional patterns of cells or tissues expressing oncogenic Ras genes, as well as upstream and downstream effectors. To validate the results obtained by array-based expression profiling, we have compared the data with those obtained by suppression subtractive hybridization and conventional expression analysis by Northern blotting. Target RNAs were prepared from preneoplastic rat ovarian surface epithelial cells (ROSE) and the KRAS-transformed derivative A2/5. By interrogating Ras signaling target arrays with mRNAs prepared from the same types of cells as hybridization target, we correctly recognized 85% of genes differentially expressed on conversion of normal ovarian epithelial cells to the Ras-transformed state.